Dpy-19 like 3-mediated C-mannosylation and expression levels of RPE-spondin in human tumor cell lines

Introduction

Glycosylation, one of the post-translational modifications, commonly regulates protein stability, folding and secretion (1–3). Certain types of glycosylation exist, including N-linked and O-linked glycosylations, C-mannosylation, and the formation of glycosylphosphatidylinositol anchors. C-mannosylation is a unique type of glycosylation in which α-D-mannose is attached directly to the indole C₂ carbon atom of a tryptophan residue via a C-C linkage (4,5). C-mannosylation occurs at the first tryptophan residue in the consensus sequence W-X-X-W/C (X represents any amino acid) (6). Certain C-mannosylated proteins, including ribonuclease 2 (7), interleukin-12 (8), properdin (9) and mindin (10), have been identified; however, the biological roles of C-mannosylation remain unclear. A previous study reported that Caenorhabditis elegans (C. elegans) dpy-19 is the C-mannosyltransferase for the thrombospondin type-1 repeat (TSR-1)-derived peptide (11) and that human dpy-19 like 3 (DPY19L3), one of the homologs of C. elegans dpy-19, catalyzes C-mannosylation of R-spondin (Rspo) 1 at the W¹⁵⁶ residue (12).

RPE-spondin (RPESP) is a protein that has unknown functions and exists in the aorta extracellular matrix (13,14). RPESP has certain domains: An N-terminal signal peptide toward the secretory pathway, followed by a somatomedin B (SMB) domain and TSR-1. Specific proteins containing the SMB domain have been reported to bind plasminogen activator inhibitor-1 and ectonucleotide pyrophosphatase/phosphodiesterase 1 (15–18); however, the physiological functions of its association remain unclear. In the TSR-1 domain, RPESP has two putative C-mannosylation sites, the W⁸⁰ and W⁸³ residues. Previously, it was demonstrated that C-mannosylation of TSR-1 in Rspo1 and Rspo3 regulates these functions (12,19). The present study focused on RPESP protein and examined the existence of C-mannosylation in RPESP. The results suggested that RPESP is C-mannosylated at both prediction sites and that DPY19L3 catalyzes C-mannosylation of RPESP at W⁸³ specifically. Therefore, it was indicated that DPY19L3 has substrate specificity and that C-mannosylation at W⁸⁰ is catalyzed by an unidentified C-mannosyltransferase. Furthermore, the present study demonstrated that the expression of RPESP was observed in certain human tumor cell lines, suggesting the malignant roles of RPESP in tumorigenesis.

Materials and methods

Cell culture

Human HT1080 (JCRB Cell Bank, Osaka, Japan) fibrosarcoma, A549 (RIKEN BioResource Center, Tsukuba, Japan) non-small-cell lung cancer, HepG2 (RIKEN...